How To Set Up A Pcr Laboratory
Setting upwardly a PCR lab from scratch
Written by Stephanie Stursberg
10. June 2022
Setting up a new lab is exciting, just can likewise be a daunting procedure. We've recently faced this challenge ourselves, and then we've put together our learnings in this five-role series. After discussing what is needed for nucleic acid extraction in part ii, we'll at present take a look at what needs to exist considered when performing polymerase chain reactions (PCRs).
Table of contents
1 How do PCR, qPCR and RT-PCR work?
Real-time and contrary transcription PCR are both ofttimes abbreviated to 'RT-PCR' but they are non the same matter.ane To make sure that nosotros're all on the same folio, we volition briefly outline the differences between these abbreviations – PCR, qPCR and RT-PCR – before exploring each technique farther:
PCR
The polymerase concatenation reaction (PCR) is a fast and cheap technique used to amplify specific Deoxyribonucleic acid segments. First, the sample is heated and then the Dna denatures (separates into ii single strands). The temperature is then lowered to let oligonucleotides to amalgamate to specific segments of the single-stranded Deoxyribonucleic acid, earlier being raised again to the optimum working temperature of the polymerase enzyme, which and then makes a complimentary re-create of the Deoxyribonucleic acid. Ane repetition or 'thermal cycle' of these steps theoretically doubles the amount of the specific Deoxyribonucleic acid present in the reaction. Depending on the amount of DNA present at the start, and the number of amplicon copies needed for post-PCR applications, 25 to 40 cycles are usually performed.
Interesting fact: The polymerase enzyme typically used in PCRs is called Taq-polymerase, because it was originally isolated from the bacterium Thermus aquaticus. Kickoff discovered in a hot leap in Yellowstone National Park in the 1960s, this enzyme can resist the loftier temperatures necessary for Deoxyribonucleic acid denaturation.
qPCR
qPCR, also called real-time PCR, quantitative PCR or quantitative real-fourth dimension PCR, is a technique used to detect and measure the amplification of target nucleic acids as they are beingness produced. In contrast to conventional PCRs, qPCRs crave a fluorescently labelled oligonucleotide probe, and a thermocycler able to measure fluorescence and calculate the bicycle threshold (CT) value. Typically, fluorescence increases in direct proportion to the concentration of the PCR product existence formed, which allows target quantities to be measured in existent-time.
RT-PCR
Reverse transcription PCR (RT-PCR) is used to amplify RNA target sequences, such as messenger RNA and viral RNA genomes. This blazon of PCR involves an initial incubation of the sample RNA with a reverse transcriptase enzyme and a Dna primer (sequence specific, oligo dT or random hexamer) before PCR amplification.
The abbreviation RT-PCR is sometimes too used for real-time PCR rather than opposite transcription PCR. We advise that real-time PCR should be abbreviated equally qPCR, since existent-time PCR is too called quantitative PCR or quantitative real-fourth dimension PCR.
PCR reactions are very sensitive and create a big number of copies of nucleic acids from minute amounts of starting material. This makes them a cardinal and highly constructive molecular biological science technique. However, because it is prone to amplicon and sample contamination, planning and designing of your PCR lab infinite will need careful consideration.
2 Designing your PCR lab
Ideally, a PCR lab should accept two rooms with ii areas, each designed for specific tasks. The first room should be exclusively used for pre-PCR activities and divided into a master mix training area and a sample preparation area. Air pressure should be slightly positive to prevent aerosols from flowing in.
The second room should have a dedicated area for nucleic acid distension, and another i for product analysis. Air pressure should be slightly negative to ensure that amplicon aerosols don't leave the room.
If you're lacking in infinite or budget for a two-room PCR lab, you can ready the pre-PCR and amplification and analysis areas in the same room, but ensure they are as far from one some other as possible.
Having pre-PCR activities spatially separated from the amplification and analysis area – either in different rooms or in separate benches – is very of import, because you lot ordinarily have a low amount of the nucleic acrid sample during preparation and a very high concentration subsequently amplification. This ways that if you clarify your PCR in the same space as y'all prepare your chief mix and samples, y'all may become false-positive results due to amplicon contagion.
You lot should besides ensure that your lab fix-up follows a unidirectional workflow. No materials or reagents used in the distension and analysis areas should ever be taken into the pre-PCR space without a thorough decontamination. This means that you'll need dedicated equipment for each surface area, e.g., two different sets of pipettes. This unidirectional workflow should also apply to lab staff. If you've been working in the amplification and analysis areas, and y'all need to go dorsum to the pre-PCR expanse, change your personal protective equipment, as it may have been contaminated by amplicon aerosols.
Another precautionary measure to take into account when setting up your PCR lab, in add-on to the spatial separation, is temporal separation. You could, for instance, consider setting up your PCR reactions in the forenoon, and perform the amplification and analysis steps in the afternoon. This may limit your flexibility, but volition preclude contagion issues and having to repeat your experiment.
3 PCR equipment tips
PCR labs typically crave a variety of equipment, such as centrifuges, vortex mixers, pipettes, fridges and freezers, thermal cyclers and assay instruments (e.g., electrophoresis systems). Depending on the size of your lab and your applications, the amount of equipment y'all'll need may vary. Instead of providing yous a 'shopping list', nosotros will outline what yous should await for when purchasing equipment and consumables in guild to keep contamination of your PCR reactions to a minimum.
Do you lot like what you're reading? Stay tuned!
3.1 Laminar flow or biosafety cabinet
Since you tin can never be 100 % certain that there are no amplicon aerosols in your pre-PCR infinite, you should set up your PCR reactions in a laminar flow hood or biosafety chiffonier, decontaminated with a bleach solution prior to starting and after y'all finish your work.
iii.ii Pipette tips and other consumables
Despite being more expensive than normal pipette tips, using filter tips for your PCR set-up will avert aerosols entering and contaminating your pipette, and avoid aerosols that might already be present in your pipette contaminating your master mix or samples. To minimize your filter tip consumption, start fill all your tubes with the primary mix using just i tip or gear up of tips – if you're using multichannel pipettes – and follow with your samples, using one tip per sample. Adding the sample last is also recommended because information technology'southward easier to dispense information technology into a liquid than into an empty tube, and because information technology reduces the take chances of aerosolizing your sample as you pipette. Acquire the best pipetting practices to assistance prevent aerosol formation.
For consumables, y'all should make certain that you lot take plenty small-scale vials available in your lab when your PCR reagents go far. Aliquoting them into smaller containers will increase their shelf life and prevent them from going through too many freeze/thaw rounds. If your reagents get contaminated, it volition too save you from throwing away your entire supply, as you lot'll have make clean aliquots available for a second PCR.
Finally, you'll demand to make sure that all consumables and equipment are costless of DNase, RNase and PCR inhibitors. Always chose sterile products from manufacturers that can certify that their tips and consumables are free of any of these potential contaminants.
iv Cleaning and contamination command
You won't need to worry about cleaning or contagion control when setting up your lab, just you volition when your lab is up and running. Nosotros will briefly address this topic below.
Whether you decide to gear up your PCR reactions in a laminar flow hood, a biosafety chiffonier or an open bench, yous volition need to decontaminate your work infinite before and afterwards set-upwards by wiping information technology with a freshly fabricated bleach solution and distilled water. The same process should be performed in the distension and assay areas. Yous should also make sure you clean your pipettes, equipment, doorknobs, and the handles of your fridges and freezers regularly.
Because PCR assays are then sensitive, all the preventative measures described hither may all the same not guarantee that your experiments will never go contaminated. It is therefore necessary to include the advisable controls to detect contagion early. Always include negative and positive controls, as this will help identifying principal mix contaminations, and ostend the functioning of the extraction protocol, reagents and amplification steps. Additionally, you should monitor the positivity rate in your lab, and ensure that unexpected increases in detection have identifiable causes, e.thou., a seasonal outbreak.
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Source: https://www.integra-biosciences.com/global/en/blog/article/setting-pcr-lab-scratch
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